A major hurdle in the studies of nitrogenase, one of the most complicated metalloenzymes known to date, is to obtain large amounts of intact, active proteins. Nitrogenase and related proteins are often multimeric and consist of metal centers that are critical for their activities. Most notably, the well-studied MoFe protein of Mo-nitrogenase is a heterotetramer that houses two of the most complicated metal clusters found in nature, the P-cluster and the FeMoco (or M-cluster). The structural complexity of these proteins and the oxygen sensitivity of their associated metal clusters, along with the demand for large amounts of high-quality proteins in most downstream analyses, make large-scale, high-yield purification of fully competent nitrogenase proteins a formidable task and yet, at the same time, a prerequisite for the success of nitrogenase research. This chapter highlights several methods that have been developed over the past few decades chiefly for the purification of naturally expressed nitrogenase in the diazotroph Azotobacter vinelandii. In addition, purification and Fe-S reconstitution strategies are also outlined for the heterologously expressed nitrogenase proteins in Escherichia coli.